High density lipoproteins (HDL) are blood-borne macromolecular complexes composed of protein and lipid that play critical roles in the prevention of cardiovascular disease (CVD), the major cause of mortality in the United States, and are under intense study as a potential enhancement to current lipid-lowering medications. These complexes are polydisperse and can contain up to 50 different proteins, each likely able to modulate the metabolism and potential benefit/pathology of the host HDL particle. Some of these proteins have surprising roles in processes such as complement regulation, protease inhibition and innate immunity. The studies in this application are aimed at understanding the molecular basis of HDL protein heterogeneity. We will test the hypothesis that HDL is composed of numerous distinct particle subpopulations, each with unique protein combinations, which play distinct physiological roles related to the protection against CVD. In Aim 1, HDL particles will be separated from normal human plasma by four orthogonal chromatographic approaches and the protein composition of fractions from each separation will be subjected to a new mass spectrometry-based pattern analysis we have developed. Protein correlations that persist across multiple separation techniques will become candidates for distinct HDL subparticles and will be verified using immunoaffinity pull-down techniques. In Aim 2, isolated HDL subfractions from Aim 1 will be subjected to homobifunctional and novel affinity-tagged cross-linking agents to covalently join closely interacting proteins to be identified by mass spectrometry. This approach will derive sequence specific structural information on the interacting proteins and serve as a complementary mode of HDL subparticle identification. We envision that this information will form a basis for future therapeutic investigations designed to specifically modulate the levels of certain cardioprotective HDL subparticles or to mimic their effects. The study may also identify HDL subspecies that can be used as biomarkers to predict protection or susceptibility to CVD. PUBLIC HEALTH RELEVANCE: High density lipoproteins (HDL) are the so-called "good cholesterol" because they protect against cardiovascular disease, but we don't understand why. The studies in this proposal will identify previously unknown HDL subspecies that may be particularly protective. This knowledge may be useful for developing predictive biomarkers and for developing preventative treatments for cardiovascular disease.